Overproduction of Chromosomal ampC β-Lactamase Gene Maintains Resistance to Cefazolin in Escherichia coli Isolates

ABSTRACT Cefazolin, an active in vitro agent against Escherichia coli, is used to treat urinary and biliary tract infections. Cefazolin is used widely as an antibiotic, and the increase in the emergence of cefazolin-resistant E. coli in many countries is a major concern. We investigated the changes in the susceptibility of E. coli clinical isolates to cefazolin following exposure. A total of 88.9% (16/18 strains) of the strains acquired resistance to cefazolin. All strains with an MIC to cefazolin of 2 μg/mL became resistant. The expression of chromosomal ampC (c-ampC) increased up to 209.1-fold in the resistant strains. Moreover, 11 of the 16 E. coli strains (68.8%) that acquired cefazolin resistance maintained the resistant phenotype after subculture in cefazolin-free medium. Therefore, the acquisition and maintenance of cefazolin resistance in E. coli strains were associated with the overexpression of c-ampC. Mutations in the c-ampC attenuator regions are likely to be maintained and are one of the key factors contributing to the increase in the number of cefazolin-resistant E. coli worldwide. IMPORTANCE This study is the first to demonstrate that mutations in the chromosomal-ampC attenuator region are responsible for the emergence of cefazolin resistance in Escherichia coli strains. The resistance was maintained even after culturing E. coli without cefazolin. This study highlights one of the key factors contributing to the increase in the number of cefazolin-resistant E. coli strains, which can pose a considerable challenge for treating common infections, such as urinary tract infections.

Authors reported acquired resistance to cefazolin in certain E. coli strains, and determined the underlying mechanism is due to the expression of chromosomal ampC. We are very curious if plasmid-borne-ampC is present or not!? This is a big problem for public health! Authors are rrequested to integrate such information or discuss it. Similar scenarios were seen with 1) chromosoamle EptA/ plasmid Mcr-1 (1 to 10) (Zhang et al., Trends Biochem Sci, 2019); 2) chromosomal TetX/ plasmid-borne Tet(X) (WIRES Mech Dis, 2022). Also, the language errors are accumulated, and recommended to revise accordingly.

Reviewer #2 (Comments for the Author):
Generally, the authors describe an interesting finding of the mechanism of cefazolin resistance in E. coli strains. However, the referee surfer from the poor presentation of results to go over the manuscript. Besides, there are still some questions needed to be interpreted in details to make the rigor of the manuscript. One of the major comment is the that sample scale is too small, only 18 strains. Furthermore, is the observation specific to cefazolin? What about other beta-lactams? Why? My detailed comments are as follows: 1. To present the results of the study more clearly, it would be better to divide the text into different parts as Introduction, Materials and methods, Results and Discussion. 2. Line 31: Specific number should be given, as "61%" is not clear enough. 3. Line 97: "Mueller-Hinton (MHB) broth" should be modified as "Mueller-Hinton broth (MHB)". 4. Line 99: Please change "day 9" to the corresponding passage times. 5. Line 100, 104, 106: Data should be shown in the corresponding figures. 6. Line 102-103: Purpose of presenting this result should be illustrated.

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RESPONSE TO REVIEWER #1
We wish to express our appreciation to Reviewer #1 for their insightful comments on our paper.
In particular, we wish to acknowledge the Reviewer's highly valuable comments regarding the presence of plasmid-borne-ampC.
Reviewer comment 1: Authors reported acquired resistance to cefazolin in certain E. coli strains, and determined the underlying mechanism is due to the expression of chromosomal ampC. We are very curious if plasmid-borne-ampC is present or not!? This is a big problem for public health! Authors are requested to integrate such information or discuss it. Similar scenarios were seen with 1) chromosoamle EptA/ plasmid Mcr-1 (1 to 10) (Zhang et al., Trends Biochem Sci, 2019); 2) chromosomal TetX/ plasmid-borne Tet(X) (WIRES Mech Dis, 2022).

Response:
We appreciate your comment on this topic. A multiplex PCR was performed for the six families of p-ampC genes (J Clin Microb, 40, 2002). We have added the following sentence at lines 141-142 (underlined): "However, none of our tested strains harbored p-ampC genes (ACC, CIT, DHA, EBC, FOX, and MOX) (J Clin Microb, 40, 2002) (data not shown)." Reviewer comment 2: Also, the language errors are accumulated, and recommended to revise accordingly.

Response:
We have proofread the English text prior to resubmission.
We have worked to incorporate your feedback and hope that the revised manuscript is now suitable for publication in your journal. We look forward to hearing from you and responding to any further questions and comments that you may have.

RESPONSE TO REVIEWER #2
Submission of revised paper #Spectrum00058-22R1 titled "Overproduction of Chromosomal ampC β-Lactamase Gene Maintains Resistance to Cefazolin in Escherichia coli Isolates." We wish to express our appreciation to Reviewer #2 for their insightful comments on our paper.
In particular, we wish to acknowledge the Reviewer's highly valuable comments regarding the mechanism of maintained cefazolin resistance in E. coli strains.
Reviewer comment: One of the major comment is the that sample scale is too small, only 18 strains. Furthermore, is the observation specific to cefazolin? What about other beta-lactams?

Why?
Response: We appreciate the Reviewer's comment on this topic. A total of 44 clinical isolates of E. coli, including E. coli ATCC 25922, were preserved in our laboratory. We selected 18 (40.9%) of the 44 strains that were resistant to cefazolin. The proportion of cefazolin-resistant E. coli has been reported as "12.1-34% in the United States, 15.2-22.3% in Australia, 63.6% in China, 39.5% in Taiwan, and 38.7% in Japan" (lines 76-78). Thus, based on the proportion, we believe it is sufficient to conclude that the overproduction of the chromosomal ampC gene maintains cefazolin resistance in E. coli isolates.
Cefazolin is often used clinically because of its antibacterial activity against E. coli. In addition, the isolation rate of resistant strains is increasing. Therefore, this study focused on revealing the mechanism of maintained cefazolin resistance.
Reviewer detailed comment 1. To present the results of the study more clearly, it would be better to divide the text into different parts as Introduction, Materials and methods, Results and Discussion.
Response: Thank you for your comments. We have written "Introduction", "Materials and methods, Results and Discussion", as per your suggestion.
Reviewer detailed comment 2. Line 31: Specific number should be given, as "61%" is not clear enough.

Response:
We have revised the following sentence at lines 30-32.
Reviewer detailed comment 4. Line 99: Please change "day 9" to the corresponding passage times.
Reviewer detailed comment 5: Line 100, 104, 106: Data should be shown in the corresponding figures.
Response: As suggested, we have expressed percentages in Figure 1. We have revised the following sentence at lines 103-105 according to Figure 1.

Reviewer detailed comment 6:
Line 102-103: Purpose of presenting this result should be illustrated.
Response: Thank you for your comments. We removed the sentence at lines 102-103 because the same content was explained at lines 110-112.
We have worked to incorporate your feedback and hope that the revised manuscript is now acceptable for publication in your journal. We look forward to hearing from you and responding to any further questions and comments that you may have. Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. The response (e.g.,: one sentence for comment 1)cannot satisfy the minimal requirement of this journal. Also, the authors fail to integrate all the relative literatures to discuss the potential significance and threat of such plasmid-borne resistance in the context of one health. Authors are suggested to compare different resistance mechansims, and extend the possible common mechanism for transmission and action.
Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary.
Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.

RESPONSE TO REVIEWER #1
Submission of the revised paper titled, "Overproduction of Chromosomal ampC β-Lactamase Gene Maintains Resistance to Cefazolin in Escherichia coli Isolates," manuscript ID: #Spectrum00058-22R2.
We wish to express our appreciation to Reviewer #1 for the insightful comments on our paper.
In particular, we wish to acknowledge the constructive suggestions, which have helped us improve the paper considerably.

Reviewer comment:
The response (e.g.,: one sentence for comment 1)cannot satisfy the minimal requirement of this journal. Also, the authors fail to integrate all the relative literatures to discuss the potential significance and threat of such plasmid-borne resistance in the context of one health. Authors are suggested to compare different resistance mechansims, and extend the possible common mechanism for transmission and action.
Response: Thank you for your comments. In response, we have included the following information in the Discussion at lines 163-171.
Line 163-171: "E. coli harboring extended-spectrum β-lactamase genes and producing AmpC Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey.
As an open-access publication, Spectrum receives no financial support from paid subscriptions and depends on authors' prompt payment of publication fees as soon as their articles are accepted. You will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.